Summary: RNA editing is an evolutionarily conserved genetic mechanism that regulates many processes, including ion channel biophysics, inflammation, energy expenditure, and skeletal muscle development. Based on these observations, we hypothesize that editing may regulate specialization of sarcomere structure and function in different skeletal muscles and thereby influence human health and disease. Nevertheless, technical constraints have heretofore prevented a systematic analysis of RNA editing in adult muscle tissues. Adar enzymes catalyze the conversion of specific adenosine residues to inosine and are thus required for the most common form of RNA editing in animals. Previous work indicates that Adar expression in skeletal muscle is inhibited by elevated expression of Aimp2; however, we note that this study used an atypical skeletal muscle as representative of the entire family of tissues. Here we demonstrate that Adar genes are expressed in all adult skeletal muscles, and that RNA editing sites are present in hundreds of key muscle genes, including structural components of the sarcomere, ion channels, and proteins involved in myokine signaling. RNA editing sites are more common in soleus than in EDL tissues, suggesting tissue-specificity in editing profiles. Consistent with this, the biased expression of the ratio of Adar:Aimp2 indicates that differences between slow- and fast-twitch muscle fibers may be due to differential RNA editing frequencies. Based on these preliminary studies, we hypothesize that RNA editing is common and physiologically significant in adult skeletal muscle. To further develop this line of investigation, we propose several specific mechanistic hypotheses, including the possibility that slow-twitch fibers have de-repressed Adar activity due to a relatively low Aimp2 expression. To test these hypotheses, we offer the following specific aims: (1) Using in silico approaches, determine whether RNA editing influences skeletal muscle specialization, (2) Using in vitro cell culture, test whether muscle-specific trans factors regulate RNA editing, and (3) Using in vivo models, test whether RNA editing regulates sarcomere function. These aims make use of the relative strengths of three independent methodologies to test the overall hypothesis that RNA editing is physiologically significant in mature skeletal muscle. Data mining will be used to compare RNA editing frequencies systematically in many different muscle tissues and disease states. Cell culture will be used to screen candidates rapidly that may regulate editing specifically in skeletal muscle. Tissue-specific mouse mutants will be used to circumvent the technical limitations of Adar knock-out mice and thereby test the effects of RNA editing in sarcomere function in adult tissues. All three aims are technically independent and mutually reinforcing. Successful completion of this research proposal will provide the first ever detailed understanding of the prevalence, significance, and regulation of RNA editing in adult skeletal muscle tissues. More broadly, it will lay the foundation for mechanistic studies into an entirely novel mechanism by which normal physiology and disease may be manipulated in skeletal muscle.